Exploring the Regulation and Function of the Human Guanine Nucleotide Exchange Factor Ect2 (Epithelial Cell Transforming Protein 2) in Cytokinesis
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Exploring the regulation and function of the human guanine nucleotide exchange factor Ect2 (epithelial cell transforming protein 2) in cytokinesis Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München Vorgelegt von Ravindra B. Chalamalasetty Martinsried / München 2006 Dissertation eingereicht am 02, February, 2006 Tag der mündlichen Prüfung : 08, March, 2006 Erstgutachter: Prof. Dr. Erich A. Nigg Zweitgutachter: Prof.Dr. Stefan Jentsch Hiermit erkläre ich, dass ich die vorliegende Dissertation selbständig und ohne unerlaubte Hilfe angefertigt habe. Sämtliche Experimente sind von mir selbst durchgeführt worden, falls nicht explizit auf dritte verwiesen wird. Ich versichere, daß ich weder versucht habe, eine Dissertation oder Teile einer Dissertation an einer anderen Stelle einzureichen, noch eine Doktorprüfung durchzuführen. München, den 24-01-2006 Table of contents Table of contents TABLE OF CONTENTS…………………………………………...….I-IV ACKNOWLEDGEMENTS SUMMARY………………………………………………………………..1 1.0 INTRODUCTION…………………………………………………….3 1.1 Overview of the cell cycle……………………………………………..3 1.1.1 An overview of mitosis………………………………………………………..4 1.1.2 An overview of cytokinesis…………………………………………………....6 1.1.3 Regulation of M phase progression…………………………………………....6 1.2 Cytokinesis…………………………………………………………….10 1.2.1 Cytokinesis in yeasts and plants……………………………………………...10 1.3 Cytokinesis in mammalian cells……………………………………...14 1.3.1 Division site determination in mammalian cells…………………………......14 1.3.2 Models for the roles of microtubules in cleavage furrow formation……........15 1.4 Central spindle and contractile ring formation…………………….20 1.4.1 The central spindle and its components…………………………...............…20 1.4.2 Cleavage furrow determination…………………………….......................…21 1.4.3 The contractile ring and formation of the cleavage furrow …………………23 1.5 Ect2........................................................................................................27 1.5.1 Ect2, a Rho family GEF required for cytokinesis……………………….......27 I Table of contents 1.5.2 Other functions of Ect2………………………………………….............…28 1.5.3 Ect2 structure: different domains of Ect2………………………….........…29 1.5.4 Regulation of Ect2 during mitosis……………………………....................32 1.6 GOAL OF MY RESEARCH............................................................34 2.0 RESULTS..........................................................................................35 2.1 Mitotic phosphorylation of human Ect2.........................................35 2.1.1 Production of polyclonal Ect2 antibodies…………………………...........35 2.1.2 Ect2 is phosphorylated during early mitosis………………………...........37 2.1.3 Identification of multiple phosphorylation sites in mitotic Ect2…….........39 2.1.4 Plk1 can phosphorylate Ect2 in vitro…………………………………......42 2.1.5 No obvious interaction between Ect2 and Plk1 kinase……………….......46 2.1.6 Analysis of Ect2 phosphorylation site mutants……………………….......47 2.1.7 Conclusion…………………………...........................................................52 2.2 Regulation of Ect2 localization.........................................................53 2.2.1 Ect2 localizes predominantly to the central spindle and cell cortex….......53 2.2.2 The amino-terminal BRCT domain is required for central spindle targeting, whereas the carboxyl-terminal PH domain targets Ect2 to the cell cortex....................................................................................................54 2.2.3 Ect2 is targeted to the central spindle via the MKlp1/MgcRacGAP and Aurora-B/MKlp2 complexes…………………………………….…….....56 2.2.4 Ect2 interacts with the MKlp1/MgcRacGAP complex via the amino- terminal BRCT domain………………………………………………......60 2.2.5 The interaction between the BRCT domain of Ect2 and the MKlp1 / MgcRacGAP might be phosphorylation dependent…………………......62 2.2.6 Ect2 central spindle localization is not essential for cytokinesis……......64 2.2.7 Conclusion………………………………………………….....................66 II Table of contents 2.3 Requirement of Ect2 in cytokinesis..................................................67 2.3.1 Ect2 controls both early and late cytokinesis events………........................67 2.3.2 RhoA and Citron kinase are not targeted to the cleavage furrow in Ect2 depleted cells……………………........................................................70 2.3.3 Nuclear targeting of the amino-terminal Ect2 (1-333) fragment can prevent cytokinesis defects…………………...........................................................73 2.3.4 The amino-terminal BRCT-containing fragment (1-333) is mislocalized as a ring-like structure perpendicular to the midbody during cytokinesis………………………………………………………………..76 2.3.5 Conclusion…………………………………………………………….......78 3.0 DISCUSSION....................................................................................79 3.1 Mitotic phosphorylation of Ect2……………………...................................79 3.2 Ect2 targeting and oncogenic potential………………………….................82 3.3 A model for Ect2 targeting to the central spindle………………………….83 3.4 Ect2 targets RhoA to the cleavage furrow independently of its central spindle localization………..........................................................................85 3.5 Interference with Ect2 function blocks cytokinesis by impairing both cleavage furrow formation and ingression………………...........................87 4.0 MATERIALS AND METHODS....................................................91 4.1 Materials………………………………………………………………......91 4.2 Plasmid constructions and site directed mutagenesis…..............................91 4.3 Antibodies………………………………………………….……………...93 4.4 Binding of antibodies to beads…………………………............................95 4.5 Generation of recombinant baculoviruses……………………….…..........96 4.6 Production of GST-Ect2 protein from sf9 insect cells…………................96 4.7 siRNA experiments……………………………………………….............97 4.8 Cell culture and generation of stable cell lines…………………...............98 4.9 Cell extracts, immunoprecipitations and western blot analysis………......98 III Table of contents 4.10 Immunoprecipitation of endogenous Ect2 from HeLa S3 spinner culture cells...............................................................................................100 4.11 Cell cycle profile and flow cytometry analysis…………………….…....101 4.12 Immunofluorescence microscopy....................................................….....101 4.13 In vitro kinase assays………………………………………………… ...102 4.14 Live-cell imaging......................................................................................103 4.15 Mass spectrometry…………………………………................................103 LIST OF ABBREVIATIONS...............................................................105 REFERENCES......................................................................................109 APPENDIX: LIST OF PLASMIDS....................................................127 RESUME...............................................................................................133 IV Acknowledgements Acknowledgements Firstly, I am thankful to Prof. Erich Nigg for giving me the opportunity to work in his department and for reviewing this manuscript. I enjoyed the opportunity of having access to scientific and technical resources within the department and the institute. I thank him for giving me the opportunity to be part of wonderful international scientific atmosphere in the department. I am also greatly indebted to Dr. Herman Silljé for being my supervisor. His continuous assistance in the form of open discussions, technical advice and brain storming exercises always encouraged me to perform more than I could possibly imagine. His guidance and suggestions are of immense help to me to learn more and carry out my PhD here. In particular, I would like to express my gratitude to Dr.Francis Barr for stimulating discussions and Dr.Roman Körner and Dr.Marjaana Nousiainen for helping and teaching me to perform MALDI-TOF mass spectrometry. My special appreciation to Alison Dalfovo for her continuous help in dealing with official documents. I would like to acknowledge Elena Nigg for technical assistance and support throughout my stay here. I would also like to express thanks to all the members of the Department of Cell Biology for their contributions and helping me to find my way and work here (special thanks to Xiumin, Xuiling, Andreas Schmidt, Stefan Huemmer, Christoph Baumann, Thomas Mayer, Claudia Szalma, Marianne Siebert and Klaus Weber for helping me in one way or the other). In addition, I would like to thank my colleagues, Herman silljé, Robert, Anja Hanisch and Jorge for critically reviewing this manuscript and giving me helpful suggestions and comments. I would like to acknowledge Anna for her advice and helping me with the formatting and organization of my thesis. I would like to thank Eunice for all her help and support in and outside the lab and for being a ‘wonderful friend’ all these years that we spent in this department and I also would like to acknowledge her for discussions on the collaborative projects. I would also like to thank Anja Wehner for friendly atmosphere at our work-bench and for helping me to pick up the stable cell line Acknowledgements clones. I am grateful to all my colleagues for the wonderful time that I spent in and outside the lab. My special thanks to Dr.Anja Schmidt from Prof.Alan Hall’s laboratory for helping me with GEF activity assays. In particular, I would like to thank Martina, Guido and Giulia for their continuous